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Method for Purifying Antigen of Foot and Mouth Disease Inactivated Virus by Hydrophobic Interaction Chromatography

A hydrophobic interaction chromatography, virus antigen technology, applied in the preparation methods of peptides, virus peptides, chemical instruments and methods, etc., can solve the problems of death, poor safety, strong side reactions, etc. The effect of high recovery rate

Active Publication Date: 2019-09-13
INST OF PROCESS ENG CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are two main problems with the foot-and-mouth vaccine: 1. The immune protection is unstable, and the antibody level is not detected or the antibody level is low after immunization, or the immunization period is short; 2. The safety is poor, and the side effects are strong. Food loss, food stoppage and even death after vaccination
These problems lead to long processing time, high material cost, and the antigen is also prone to lose immunological activity during the long-term processing

Method used

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  • Method for Purifying Antigen of Foot and Mouth Disease Inactivated Virus by Hydrophobic Interaction Chromatography
  • Method for Purifying Antigen of Foot and Mouth Disease Inactivated Virus by Hydrophobic Interaction Chromatography
  • Method for Purifying Antigen of Foot and Mouth Disease Inactivated Virus by Hydrophobic Interaction Chromatography

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Get 1000mL of the cell culture supernatant containing FMD virus, the total protein concentration is 0.47g / L, and the FMD virus antigen concentration is 2.1 μg / mL.

[0029] Use a plate ultrafiltration membrane (Sartorius) with a molecular weight cut-off of 300kDa to concentrate the cell supernatant to about 20mL once, and the protein concentration in the sample is about 100μg / mL; the membrane flow rate during ultrafiltration is 100cm / s, and the pressure is controlled below 0.3MPa .

[0030] In the supernatant, adjust the conductivity and pH to above 100mS / cm and pH 8.0 respectively with ammonium sulfate and sodium hydroxide solution in turn, and then feed into the sodium phosphate buffer that has been adjusted to conductance 100mS / cm with ammonium sulfate in advance (pH8.0) balanced Butyl Sepharose 4FF hydrophobic chromatography column (GE Healthcare, 5cm×1.6cm I.D.), after feeding, after continuous elution, ammonium sulfate was used to adjust to sodium phosphate buffer ...

Embodiment 2

[0034] Get 100mL of cell culture supernatant containing FMD virus, the total protein concentration is 0.47g / L, and the FMD virus antigen concentration is 2.1 μg / mL.

[0035]In the supernatant, adjust the conductivity and pH to above 100mS / cm and pH 7.2 respectively with ammonium sulfate and sodium hydroxide solution in turn, then feed into the sodium phosphate buffer that has previously adjusted the conductivity to 100mS / cm with ammonium sulfate (pH7.2) balanced Butyl Sepharose 4FF hydrophobic chromatography column (GE Healthcare, 5cm×1.6cm I.D.), after feeding and continuing to rinse, use ammonium sulfate to adjust the conductivity to 5-100mS / cm sodium phosphate buffer solution (pH 7.2) to carry out gradient elution, and collect the eluate containing the ultraviolet absorption peak whose recovery rate of foot-and-mouth disease virus antigen is greater than 20%. The chromatography medium was regenerated with 1M sodium hydroxide solution.

[0036] The antigen concentration and...

Embodiment 3

[0038] Get 100mL of cell culture supernatant containing FMD virus, the total protein concentration is 0.47g / L, and the FMD virus antigen concentration is 2.1 μg / mL.

[0039] Concentrate the cell supernatant to about 10 mL at a time using a plate-type ultrafiltration membrane (Sartorius) with a molecular weight cut-off of 50 kDa, and the concentration of FMD virus antigen in the sample is about 20 μg / mL; the membrane flow rate during ultrafiltration is 100 cm / s, and the pressure is controlled at 0.3 Below MPa.

[0040] In the supernatant, use ammonium sulfate and sodium hydroxide solution to adjust the conductivity and pH to more than 50mS / cm and about pH 9.0 respectively, then add 10ml of sodium phosphate buffer ( pH 9.0) balanced Phenyl Sepharose 6FF hydrophobic medium, stirred and adsorbed at 100-200rpm for 30min, then added to a hydrophobic chromatography column (GE Healthcare, 5cm×1.6cm I.D.), after continuous elution, adjusted with ammonium sulfate to The sodium phosphat...

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Abstract

The invention relates to a method for purifying a foot-and-mouth disease inactive virus antigen from a cell culture liquid. The method is characterized in that the foot-and-mouth disease inactive virus antigen is adsorbed on a chromatographic column through interaction between a hydrophobic group on the surface of a foot-and-mouth disease virus and a hydrophobic group between a hydrophobic action chromatographic packing, so the foot-and-mouth disease inactive virus antigen is separated from impurities in the liquid. The foot-and-mouth disease inactive virus antigen adsorbed on the chromatographic column can be eluted under mild eluting conditions in order to realize the purifying purpose. The method has the advantages of fast speed, few operation steps, stable process, high yield of the foot-and-mouth disease inactive virus antigen, easy amplification and industrial scale production, and large practical application values.

Description

technical field [0001] The invention relates to the field of separation and purification of vaccine antigens, in particular to a chromatographic separation method for separation and purification of foot-and-mouth disease inactivated virus vaccine antigens from cell culture fluid. Background technique [0002] Foot and Mouth Disease (FMD) is a severe infectious disease caused by Foot-and-Mouth Disease Virus (FMDV). It mainly infects cloven-hoofed animals such as cattle, sheep, and pigs. Infection, but the symptoms are relatively mild. Foot-and-mouth disease epidemics often bring huge losses to the livestock industry. Due to its strong infectivity, fast transmission speed and great harm, foot-and-mouth disease has been the focus of international health circles since the beginning of the 20th century. Countries have established research institutions to find ways to control foot-and-mouth disease. It was not until the end of the 19th century that the foot-and-mouth disease vir...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/09C07K1/20
Inventor 苏志国张松平杨延丽马光辉李浩张焱
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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